Dept. of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University, Rome, Italy.
J Biol Regul Homeost Agents. 2011 Apr-Jun;25(2 Suppl):S43-51.
Many studies demonstrated that human adult cardiac progenitor cells in the form of cardiospheres (CSps) could represent a powerful candidate for cardiac cell therapy. To achieve the clinical translation of this biotechnological product, the development of well-defined culture conditions is required to optimize their proliferation and differentiation. Thrombin, a serine protease acting through the protease-activated receptor 1 (PAR-1) signalling to modulate many cellular functions such as proliferation and differentiation in several cell types, is one of the factors included in the CSps medium. Therefore, the assessment of the effective dependence of the thrombin related cellular effects from PAR-signalling is strategic both for understanding the biological potential of these cells and for the GMP translation of the medium formulation, using synthesised analogs. In this study the effects of thrombin on human CSps and their potential relationship with the specific proteolytic activation of PAR-1 have been investigated in different culture conditions, including thrombin inhibitor hirudin and PAR-1 agonist/ antagonist peptides TFLLR and MUMB2. In this study we show that, in the presence of thrombin and TFLLR, CSps, in which PAR-1 expression was evidenced by immunofluorescence and western blot analysis, increase their proliferation activity (BrdU assay). Such increased proliferative rate was consistently associated with a higher phosphorylation level of the cell cycle inhibitor GSK3. Concerning the assessment of the potential effects of thrombin and its agonist on differentiation, both western blot and real-time PCR analysis for stemness, cardiac and vascular markers (such as cKit, cx43 and KDR) showed that CSps commitment was substantially unaffected, except for GATA4 mRNA, whose transcription was down-regulated in the presence of the natural protease, but not after treatment with TFLLR. In conclusion, activation of PAR-1-dependent signalling is important to support CSps proliferative potential, keeping unaltered or at best stable their differentiation properties. The availability of thrombin agonists, such as TFLLR, able to guarantee the required growth effect without affecting CSps lineage commitment, could represent a technological improvement for cost-effective, easy-to-handle and GMPtranslatable synthetic media.
许多研究表明，以心脏球（CSps）形式存在的人类成体心脏祖细胞可以成为心脏细胞治疗的有力候选物。为了实现这种生物技术产品的临床转化，需要开发明确的培养条件来优化其增殖和分化。凝血酶是一种丝氨酸蛋白酶，通过蛋白酶激活受体 1（PAR-1）信号传导来调节多种细胞功能，如增殖和分化，是 CSps 培养基中的一种因子。因此，评估凝血酶相关细胞效应与 PAR 信号之间的有效依赖性对于理解这些细胞的生物学潜力以及使用合成类似物进行培养基配方的 GMP 转化都具有战略意义。在这项研究中，在不同的培养条件下，包括凝血酶抑制剂水蛭素和 PAR-1 激动剂/拮抗剂肽 TFLLR 和 MUMB2，研究了凝血酶对人 CSps 的影响及其与 PAR-1 特异性蛋白水解激活的潜在关系。在这项研究中，我们表明，在凝血酶和 TFLLR 的存在下，CSps 通过免疫荧光和 Western blot 分析证实 PAR-1 的表达，增加了它们的增殖活性（BrdU 测定）。这种增殖率的增加与细胞周期抑制剂 GSK3 的磷酸化水平升高一致。关于评估凝血酶及其激动剂对分化的潜在影响，Western blot 和实时 PCR 分析用于干细胞、心脏和血管标志物（如 cKit、cx43 和 KDR）表明，CSps 的分化基本上没有受到影响，除了 GATA4 mRNA，其转录在天然蛋白酶存在下下调，但在用 TFLLR 处理后没有下调。总之，PAR-1 依赖性信号的激活对于支持 CSps 的增殖潜力很重要，同时保持其分化特性不变或最好稳定。凝血酶激动剂（如 TFLLR）的可用性能够保证所需的生长效果，而不会影响 CSps 谱系的分化，这可能代表着一种技术改进，可用于经济高效、易于处理和符合 GMP 要求的合成培养基。
